In this proposal, we will employ X-ray crystallography as the chief tool to study the structures and mechanisms of two medically-essential enzymes: taurine/alpha-ketoglutarate dioxygenase (TauD)from Escherichia coli and S-(2)-hydroxypropylphosphonic acid epoxidase (HppE)from Streptomyces wedmorensis. Both enzymes belong to the non-heme mononuclear-iron dependent protein family, which activates oxygen to carry difficult reactions. These reactions are essential for critical functions such as oxygen sensing; hypoxia response; herbicide degradation; fatty acid, collagen, and antibiotic synthesis; and repair of alkylated DNA. TauD, the prototype of the Fe(ll)-alpha-ketoglutarate (aKG) dependent oxygenases, catalyzes the hydroxylation of taurine to aminoacetaldehyde and sulfite, concurrent with the conversion of aKG to succinate and carbon dioxide, to provide sulfur to the bacterium. The mononuclear iron enzyme, HppE, catalyzes the formation of the antibiotic fosfomycin, an unusual C-P-bond-containing epoxide, from S-(2)-hydroxypropylphosphonic acid (S-HPP). The reaction is a two-electron oxidation and is mechanistically atypical because it is independent of aKG or any other cofactor and results in incorporation of the hydroxyl oxygen of the substrate, rather than an atom of dioxygen, into the epoxide ring. Our studies will employ mechanistic crystallography to unravel vague details in the well-studied oxygen activation reactions of TauD and HppE. We will solve the structures of the nitric oxide (a catalytically inert mimic of dioxygen) adducts of TauL>Fe(ll)*aKG*taurine (or ta urine analogues (1,1-dideuterotaurine or 1,1- difluorotaurine)) and HPPE"Fe(ll)"S-HPP to address binding modes of oxygen to the metal center, accomanying conformational dynamics at the active site, and the nature of the reaction steps that follow. We will also determine the structures of the HppE-Fe(ll)-nitrosyl complex with R-(2)- hydroxypropylphosphonic acid (R-HPP) and S-(2)-hydroxy-(2)-phenylethylphosphonic acid (S-HPEP) to understand the enzyme's regio- and stereo-specificity. Relevance to Public Health: Insight obtained from our studies into the structures and mechanisms of TauD and HppE will contribute to the advancement of clinical research on several disease states, such as cancer, alcoholic liver cirrhosis, Ehlers-Danlos syndrome, and fibrotic disease, which are associated with dysfunction in a number of enzymes in this family of proteins. [unreadable] [unreadable] [unreadable]